Free-flowing lyophilized tobramycin formulation

ABSTRACT

The invention provides among other things a stable, sterile pharmaceutical formulation comprising lyophilized tobramycin, wherein the lyophilized tobramycin is in the form of a free-flowing powder. The invention also provides a method of producing a stable, sterile pharmaceutical product comprising lyophilized tobramycin. The invention also provides a pharmaceutical dosage form comprising the pharmaceutical formulation, as well as a method of treating a disease in a patient comprising administering a solution of the pharmaceutical formulation to a patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/827,024 filed Apr. 19, 2004.

FIELD OF THE INVENTION

This invention pertains among other things to lyophilized tobramycin,solutions thereof, and methods of preparing and using lyophilizedtobramycin.

BACKGROUND OF THE INVENTION

Tobramycin sulfate is an aminoglycoside antibiotic derived fromStreptomyces tenebrarius which has the formula:

Tobramycin sulfate is chemically described as0-3-amino-3-deoxy-α-D-glucopyranosyl-(1→4)—O—[2,6-diamino-2,3,6-trideoxy-α-D-ribo-hexopyranosyl-(1→6)]-2-deoxy-L-streptamine,sulfate (2:5)(salt). Tobramycin sulfate has the molecular formula(C₁₈H₃₇N₅O₉)₂.5H₂SO₄, and has a molecular weight of 1425.45.

Tobramycin sulfate is approved in the United States for the treatment ofthe following infections when caused by susceptible organisms:septicemia, complicated and recurrent urinary tract infections, lowerrespiratory infections, serious skin and soft tissue infectionsincluding burns and peritonitis, and central nervous system (CNS)infections caused by organisms resistant to other antibiotics.Tobramycin sulfate is typically active against most strains of thefollowing organisms in vitro and in clinical infections: P. aeruginosa,Proteus sp. (indole-positive and indole-negative), including P.mirabilis, M. morganii, P. rettgeri, and P. vulgaris, E. coli,Klebsiella-Enterobacter-Serratia group, Citrobacter sp., Providenciasp., Staphylococci, including S. aureus (coagulase-positive andcoagulase-negative).

Tobramycin sulfate is currently sold in the U.S. as a sterile dry powderin 1.2 g Pharmacy Bulk Package dose vials (e.g., marketed by Eli Lillyand Company under the tradename Nebcin®). For therapeutic applications,the powder is dissolved in a sterile aqueous vehicle such as SterileWater for Injection, USP, and is administered by injection. Typically,the 1.2 g dosage form is constituted in 30 mL of the aqueous vehicle toachieve a final concentration of 40 mg/mL. Solutions of tobramycinsulfate may be diluted further in injectable fluids such as 5% Dextrosein water, USP, and 0.9% Sodium Chloride Injection, USP. Optionally, thetobramycin formulation is first constituted (e.g., with sterile water)and then further diluted (e.g., with a sodium chloride solution).

Tobramycin sulfate is normally administered via intramuscular (i.m.) orintravenous (i.v.) injection at a dosage between about 2 mg/kg per dayto about 5 mg/kg per day for adults, depending on the infection to betreated. Tobramycin sulfate is normally administered to children at adosage of 6 to 7.5 mg/kg per day, and is administered to neonates at adosage up to 4 mg/kg per day. The duration of treatment typically isseven to ten days. After reconstitution, e.g., with 30 mL of SterileWater for Injection, tobramycin solutions should be used within 24 hoursif stored at room temperature, or within 96 hours if stored underrefrigeration.

The manufacture of tobramycin sterile powder involves a powder filloperation that presents a number of practical problems. During theproduction of the sterile powder, the powder is processed in a way thatcauses variations in the flow properties. The variation in flowproperties greatly impairs the accuracy of dosage during the fillingprocess. The sterile powder fill operation further employs specializedequipment. The powder fill operation is costly and may be furthercomplicated by the risk of microbial contamination during the operation.Moreover, the powder fill process creates risks of accidental exposureto the powder by personnel that are involved in the production process.

Approaches for manufacturing lyophilized forms of tobramycin sulfatehave been proposed including tobramycin for injection marketed byPharma-Tek Inc. While this lyophilization process overcomes some of theproblems associated with the crystalline powder, it produces a cake inwhich the lyophilizate particles adhere to each other and to the wallsof its container.

A method for freeze drying tobramycin sulfate to produce a friablefreeze dried powder has been developed that entails use of atert-butanol/water cosolvent system (Nail et al., J. Pharm. Sciences,91:1147-1155 (2002). While this method produces a loose powder that“readily breaks apart”, the method disclosed is not adapted for thecommercial production of tobramycin sulfate.

Thus, there remains a need for a stable, sterile form of lyophilizedtobramycin having a smooth, uniform cake and non-adherent particles,methods of producing such a formulation that are appropriate forcommercial scale, and methods of using such a formulation. The inventionprovides such a formulation and methods. These and other advantages ofthe present invention, as well as additional inventive features, will beapparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides among other things a stable, sterilepharmaceutical formulation comprising lyophilized tobramycin, whereinthe lyophilized tobramycin is in the form of a free-flowing powder. Theinvention also provides a solution prepared by dissolving thepharmaceutical formulation in an aqueous vehicle. The invention furtherprovides a liquid composition comprising tobramycin (e.g., tobramycinsulfate) and a solvent which comprises tert-butyl alcohol.

The invention also provides a method of producing a stable, sterilepharmaceutical product comprising lyophilized tobramycin, which methodcomprises preparing a composition comprising tobramycin (e.g.,tobramycin sulfate) and a solvent, which solvent comprises tert-butylalcohol, and lyophilizing the composition, wherein the lyophilizedtobramycin is in the form of a free-flowing powder.

The invention further provides a method of producing a stable, sterilepharmaceutical formulation comprising lyophilized tobramycin, whichmethod comprises (a) preparing a liquid composition comprisingtobramycin (e.g., tobramycin sulfate) and a solvent which comprisestert-butyl alcohol, (b) freezing the composition to a temperature offrom about −10° C. to about −70° C., to produce a frozen mixture,wherein the temperature is maintained for from at least about 1 hour toabout 30 hours, (c) subjecting the frozen mixture to a primary dryingstage, which comprises applying a vacuum to reduce the pressure by anamount effective to remove aqueous solvent from the frozen mixture, and,while applying the vacuum, changing the temperature of the frozenmixture to a primary drying temperature, wherein the primary dryingtemperature is from about −15° C. to about 20° C., and wherein theprimary drying temperature is maintained for at least about 40 hours toabout 80 hours, to produce a first intermediate, and (d) subjecting thefirst intermediate to a secondary drying stage, which comprises applyinga vacuum to reduce the pressure by an amount effective to remove aqueoussolvent from the first intermediate, and, while applying the vacuum,changing the temperature of the frozen mixture to a secondary dryingtemperature, wherein the secondary drying temperature is from about 30°C. to about 45° C., and wherein the secondary drying temperature ismaintained for at least about 15 hours to about 30 hours.

The invention additionally provides a pharmaceutical dosage formcomprising a sealed container and a pharmaceutical formulationcomprising a therapeutically effective amount of lyophilized tobramycincontained within the container. The invention provides a method oftreating a disease in a patient, which comprises dissolving theabove-described pharmaceutical formulation in a pharmaceuticallyacceptable solvent to produce a pharmaceutically acceptable solution,and administering the solution to a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides among other things a stable, sterilepharmaceutical formulation comprising lyophilized tobramycin, whereinthe lyophilized tobramycin is in the form of a free-flowing powder. Thelyophilized tobramycin of the present invention is a white to off-whitesolid of high purity. The lyophilized tobramycin of the presentinvention preferably has a purity of greater than about 90% (i.e.,contains about 10% or less of total impurities based on the total weightof tobramycin), more preferably has a purity of about 96% or greater(i.e., contains about 4% or less of total impurities based on the totalweight of tobramycin), and even more preferably has a purity of about98% or greater (i.e., contains about 2% or less of total impuritiesbased on the total weight of tobramycin). Most preferably, thelyophilized tobramycin has a purity of about 98%, about 98.5%, or about99% (i.e., contains about 2%, about 1.5%, or about 1.0%, respectively,of total impurities based on the total weight of tobramycin). Purity canbe assessed based on potency. Purity also can be determined by highperformance liquid chromatography assay (e.g., allowing separation ofpure lyophilized tobramycin from impurities, and quantitation of therelative amounts by the determination of the peak area of puretobramycin and/or the impurities area as compared to the total peakarea), or by a similar method.

The lyophilized tobramycin formulation can comprise any suitable amountof tobramycin, but preferably comprises a therapeutically effectiveamount of tobramycin. A “therapeutically effective amount” means anamount sufficient to show a meaningful benefit in an individual, e.g.,promoting at least one aspect of antimicrobial activity, or treatment,healing, prevention, or amelioration of other relevant medicalcondition(s) such as that associated with a particular microbialinfection. Therapeutically effective amounts may vary depending upon thebiological effect desired in the individual, condition to be treated,and the individual. In this regard, the lyophilized tobramycinpreferably is present in the formulation in an amount from about 0.5grams to about 5.0 grams (e.g., about 1.0 g, about 1.5 g, about 2.0 g,about 2.5 g, about 3.0 g, about 3.5 g, about 4.0 g, about 4.5 g, orabout 5.0 g). Most preferably, the lyophilized tobramycin is present inan amount of from about 0.5 g to about 1.5 g, especially in an amount ofabout 1.2 g.

The inventive lyophilized tobramycin preferably is in the form of afree-flowing powder. The term “free-flowing powder,” as used herein,refers to a powder that shows minimal or no compaction, and whichcontains particles that adhere minimally or not at all to each other orto surfaces with which the particles are in contact (e.g., containerwalls). In the event that minimal compaction or adherence is observed,the powder according to the invention is such that the minimallycompacted or adhered powder readily breaks up into a free-flowing formwith gentle tapping or shaking of the vessel containing the powder. Theflowability of a powder is affected by a variety of factors, includingphysical characteristics of the particles themselves (e.g., size, shape,angularity, and hardness), as well as external factors such as humidity,vibration, and aeration. The flow properties (also referred to asrheological properties) of a powder can be measured using any suitabletechnique known in the art including, for example, measuring the angleof repose of the powder and timing the flow of a powder through anaperture. Alternatively, a powder rheometer can be used to determineflowability.

The lyophilized tobramycin formulation can be prepared using anysuitable form of tobramycin, including in particular tobramycin base(i.e.,0-3-amino-3-deoxy-α-D-glucopyranosyl-(1->6)-O-(2,6-diamino-2,3,6-trideoxy-α-D-ribo-hexopyranosyl-(1->4)-2-deoxy-d-streptamine))and tobramycin salts, especially tobramycin sulfate. Optionally, ratherthan a tobramycin sulfate salt, another tobramycin salt can be usedinstead in the formulations of the invention, e.g., a sugar acid salt(e.g., as described in U.S. Pat. Nos. 5,595,977 and 6,077,822), anitrate salt (e.g., as described in U.S. Patent Application2003/0105066), and the sparingly-soluble tobramycin salts tobramycinlaurate, tobramycin myristate, and tobramycin dodecyl sulfate (e.g., asdescribed in U.S. Patent Application 2004/0048786). A preferred form oftobramycin according to the invention for preparation of the lyophilizedtobramycin formulation is tobramycin sulfate.

In a preferred embodiment of the invention, the lyophilized tobramycinformulation has a low moisture content. The moisture content of theinventive lyophilized tobramycin is the result of residual aqueoussolvent (i.e., water) that remains in the formulation after thelyophilization process. Additionally, some small amount of organicsolvent optionally remains in the formulation following lyophilization.The organic solvent content and/or the moisture content can be theproduct of any suitable solvent that is used in the method of producingthe lyophilized tobramycin formulation described herein. Suitablesolvents include, for example, aqueous solvents (i.e., water, whichcould contribute to the moisture content), organic solvents (which couldcontribute to the organic solvent content), or a combination of anaqueous solvent and an organic solvent (which could contribute to boththe moisture content and the organic solvent content). Preferably, thesolvent according to the invention comprises water and an organicsolvent. Suitable organic solvents include, for example, alcohols (e.g.,ethanol, isopropyl alcohol, and tert-butyl alcohol (TBA)). Morepreferably, the organic solvent is a sterically hindered alcohol, suchas tert-butyl alcohol; however, any suitable organic solvent can be usedin the invention. Most preferably, the organic solvent is tert-butylalcohol. In a preferred embodiment, the solvent according to theinvention preferably comprises tert-butyl alcohol and water. Themoisture content of the inventive lyophilized tobramycin formulationdesirably is measured as the content of water present in the lyophilizedtobramycin formulation, whereas the organic solvent content of theinventive lyophilized tobramycin formulation desirably is measured asthe content of organic solvent present in the lyophilized tobramycinformulation. In this regard, the lyophilized tobramycin preferablycontains less than about 2 wt % of water (where the wt % is the % waterrelative to the total weight of the lyophilized tobramycin formulation).The lyophilized tobramycin preferably contains less than about 1.1 wt %of organic solvent such as tert-butyl alcohol (where the wt % is the %organic solvent relative to the total weight of the lyophilizedtobramycin formulation.). More preferably, the lyophilized tobramycinsulfate contains about 1 wt %, about 0.9 wt %, about 0.8 wt %, or about0.7 wt % of an organic solvent such as tert-butyl alcohol. When anorganic solvent other than tert-butyl alcohol is employed, the organicsolvent wt % can be adjusted accordingly, e.g., based on safety and/ortoxicity concerns.

The components of the lyophilized tobramycin formulation of theinvention (e.g., the tobramycin and the organic solvent) are furtherdescribed below in the context of the liquid composition. The “liquidcomposition” is the formulation of the invention prior to lyophilization(e.g., the pre-lyophilization solution). While the pre-lyophilizationsolution may not be optimal for a pharmaceutical formulation, in someinstances, however, it may be desirable to constitute (and optionallyfurther dilute) a pharmaceutical lyophilized tobramycin formulation foruse as a liquid (e.g., solution). Along these lines, a pharmaceuticalformulation according to the invention comprises tobramycin (e.g.,tobramycin sulfate) and solvent (as well as further optional components)preferably either in lyophilized form, or as a liquid. For the liquidcompositions of the invention to serve as pharmaceutical tobramycinformulations, optionally additional components are included, such aspreservatives (e.g., methylparaben and propylparaben), stabilizers(e.g., sugars), and buffering agents (e.g., sodium citrate, sodiumhydroxide, sodium acetate, and others).

The inventive lyophilized tobramycin formulation can be contained withina sealed container. Preferably, the inventive lyophilized tobramycinformulation is contained within a container that is sealed aseptically.More preferably, the container is provided with an opening and a meansfor aseptically sealing the opening, e.g., such that the sealedcontainer is fluidly sealed or the sealed opening is substantiallyimpermeable to atmospheric gasses, moisture, pathogenic microorganisms,or the like. The container can be constructed of any suitable materialsuch as, for example, glass, polypropylene, Dalkyo Resin CZ (sold byDalkyo Gomu Seiko, Ltd.), polyethylene terephthalate, and the like. In apreferred embodiment, the container is constructed of glass. Suitableglass containers include, but are not limited to, glass vials. Suitableglass vials include molded and tubing glass vials such as, for example,Type I molded glass vials, and the like. Such molded and tubing glassvials can be obtained from Kimble Glass, Inc., Vineland, N.J., WheatonScience Products, Millville, N.J., or other companies.

A suitable means for sealing the container can include, for example, astopper, a cap, a lid, a closure, a covering which fluidly seals thecontainer, or the like. Examples of suitable closures include closuresthat are suitable for medical vials, such as those described in U.S.Pat. No. 4,671,331, and references cited therein. The means for sealingthe container are not limited to separate closures or closure devices.In a preferred embodiment, the means for aseptically sealing thecontainer includes a stopper such as, for example, a stopper that isconfigured to fluidly seal the opening. Suitable stoppers includeconventional medical grade stoppers which do not degrade or releasesignificant amounts of impurities upon exposure to the constitutedaqueous tobramycin solution. Preferably, the stopper is constructed ofan elastomer, which is more preferably an elastomer that is pierceableby a hypodermic needle or a blunt cannula. Exemplary stoppers include6720 GC gray rubber stoppers from American Stelmi Corporation, 4432/50gray rubber stoppers from West Company, and the like.

Optionally, an outer seal is provided which covers and entirelysurrounds the stopper. The outer seal can be constructed of any suitablematerial. When an outer seal is used, it is preferably fitted with a lidthat can be easily manually removed to provide access to the stopper.Suitable outer seals can include, for example, Flip-offAluminum/Polypropylene Seals (lacquered or non-lacquered aluminum),marketed by The West Company, Inc., and other manufacturers. Such sealsinclude an outer rim made of a suitable material, such as aluminum, thatentirely surrounds the lateral edge of the stopper and further include alid (typically polypropylene or other suitable material) that entirelycovers the upper surface of the stopper. The polypropylene lid can be“flipped” off e.g., by exerting upward pressure with a finger or thumb,to provide access to the stopper, e.g., so that it can be punctured witha hypodermic needle to deliver an aqueous vehicle for constitution (see,e.g., U.S. Pat. No. 6,136,814). Optionally, the seal can be removed inits entirety to allow the powder to be poured from the vial.

Preferably, the container contains a therapeutically effective dose ofthe inventive lyophilized tobramycin formulation and is of sufficientvolume (i.e., has sufficient capacity) to contain the volume of solutionthat is recommended for constitution. More preferably, the containercontains tobramycin in an amount which is an approved dosage fortreating microbial infections, such as those described herein, and is ofsufficient volume (i.e., has sufficient capacity) to contain the totalvolume of solution recommended for constitution. In a particularlypreferred embodiment, the container volume is about 50 mL, and about 1.2g of the inventive lyophilized tobramycin formulation are containedwithin the container.

The invention further provides a solution prepared by dissolving theinventive lyophilized tobramycin formulation in an aqueous vehicle. Theaqueous vehicle is preferably a sterile aqueous vehicle that is normallyused as liquid vehicle for injection. Suitable aqueous vehicles include,for example, sterile water (e.g., Sterile Water for Injection, USP),sodium chloride solutions (e.g., 0.9% Sodium Chloride Injection, USP),dextrose solutions (e.g., 5% or 10% Dextrose for Injection), sodiumchloride/dextrose mixtures (e.g., 5% Dextrose and 0.225% Sodium Chloridefor Injection, 5% Dextrose and 0.45% Sodium Chloride for Injection),Lactated Ringer's for Injection, and mixtures thereof. Optionally, thelyophilized tobramycin formulation is first reconstituted (e.g., withsterile water) and then further diluted (e.g., with a sodium chloridesolution).

The inventive lyophilized tobramycin formulation can be dissolved in anysuitable volume of the aqueous vehicle. Preferably, the lyophilizedtobramycin formulation is dissolved in about 50 mL or less of vehicle(e.g., about 40 mL, about 30 mL, about 20 mL, about 10 mL, or about 5mL). Preferably, the lyophilized tobramycin formulation is dissolved inthe aqueous vehicle, such that the final concentration of tobramycin inthe solution is about 40 mg/mL or less (e.g., is about 40 mg/mL, about35 mg/mL, about 30 mg/mL, about 25 mg/mL, about 20 mg/mL, about 15mg/mL, or 10 mg/mL, about 5 mg/mL). Preferably, after further dilutionthe final concentration of tobramycin in the solution is about 2 mg/mLor less (e.g., about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about0.4 mg/mL, about 0.3 mg/mL, or about 0.2 mg/mL). Most preferably, thefinal concentration of tobramycin in the solution is from about 0.2mg/mL to about 2 mg/mL.

As described above, the invention also provides a liquid compositioncomprising tobramycin (e.g., tobramycin sulfate) and a solvent, whichsolvent comprises tert-butyl alcohol. The liquid composition cancomprise any suitable amount of tert-butyl alcohol, so long as, uponlyophilization of the liquid composition, the tobramycin is in the formof a free-flowing powder. In this regard, the liquid compositionpreferably contains from about 1.5% to about 9.0% by volume (v/v) oftert-butyl alcohol (e.g., about 9.0% v/v, about 8.5% v/v, about 8.0%v/v, about 7.5% v/v, about 6.5% v/v, about 5.5% v/v, about 4.5% v/v,about 3.5% v/v, about 2.5% v/v, or about 1.5% v/v). Most preferably, theliquid composition comprises about 4.5% by volume or less (e.g., about4% v/v, about 3.5% v/v, about 3% v/v, about 2% v/v, about 1% v/v, orabout 0.5% v/v) of tert-butyl alcohol.

The liquid composition can comprise any suitable amount of tobramycin.In a preferred embodiment of the invention, the tobramycin (e.g.,tobramycin sulfate) is present in the liquid composition in an amount offrom about 60 mg/mL to about 100 mg/mL. In a particularly preferredembodiment of the invention, the tobramycin (e.g., tobramycin sulfate)is present in the liquid composition in an amount of from about 70 mg/mLto about 90 mg/mL (e.g., about 70 mg/mL, about 75 mg/mL, about 80 mg/mL,about 85 mg/mL, or about 90 mg/mL). Most preferably, the tobramycin(e.g., tobramycin sulfate) in the liquid composition is present in anamount of about 83.2 mg/mL. One of ordinary skill in the art would knowhow to modify the composition for use of forms other than tobramycinsulfate.

In addition, the solvent of the liquid composition preferably containssterile water, such as Sterile Water for Injection, USP.

The inventive liquid composition can further comprise excipients thatare routinely employed in lyophilization formulations. Such excipientsinclude, for example, buffering agents, surfactants, cryoprotectants,and bulking agents. Mannitol, for example, typically is used in the artas an excipient in lyophilization formulations. However, other suitableexcipients can be included which preferably do not deleteriously impactthe properties of the inventive liquid composition. Examples of suchexcipients (e.g., buffering agents) include sodium or potassiumphosphate, citric acid, lactic acid, tartaric acid, gelatin, glycine,and carbohydrates such as lactose, maltose, dextrose, dextran,hetastarch, and the like. The excipients can be used alone or incombination to provide a cake of good quality which readily disperses inan aqueous vehicle upon reconstitution.

The invention provides a method of producing a stable, sterilepharmaceutical product comprising lyophilized tobramycin, which methodcomprises preparing a composition comprising tobramycin (e.g.,tobramycin sulfate) and a solvent, which solvent comprises tert-butylalcohol, and lyophilizing the composition, wherein the lyophilizedtobramycin is in the form of a free-flowing powder.

The invention also provides a method for producing the inventivelyophilized tobramycin formulation, which comprises (a) preparing acomposition comprising tobramycin (e.g., tobramycin sulfate) and asolvent which comprises tert-butyl alcohol, (b) freezing the compositionto a temperature of from about −10° C. to about −70° C., to produce afrozen mixture, (c) subjecting the frozen mixture to a primary dryingstage, which comprises applying a vacuum to reduce the pressure by anamount effective to remove the solvent from the frozen mixture, and,while applying the vacuum, changing (e.g., raising) the temperature to aprimary drying temperature, wherein the primary drying temperature isfrom about −15° C. to about 20° C., to produce a first intermediate, and(d) subjecting the first intermediate to a secondary drying stage, whichcomprises applying a vacuum to reduce the pressure by an amounteffective to remove the solvent from the first intermediate, and, whileapplying the vacuum, changing (e.g., raising) the temperature to asecondary drying temperature, wherein the secondary drying temperatureis from about 30° C. to about 45° C., to produce the pharmaceuticalformulation. Descriptions of the tobramycin (e.g., tobramycin sulfate)and the solvent comprising tert-butyl alcohol, and components thereof,set forth above in connection with the inventive pharmaceuticalformulation and liquid composition, also are applicable to those sameaspects of the inventive method.

The composition is “frozen” or cooled to a temperature that freezes thetert-butyl alcohol solvent. Preferably, the liquid composition is frozensufficiently to allow for its removal under reduced pressure (e.g., bysublimation). Desirably, the liquid composition is frozen to atemperature of about −10° C. or lower (e.g., from about −10° C. to about−70° C., from about −20° C. to about −70° C., from about −30° C. toabout −70° C., or from about −30° C. to about −60° C.), and ispreferably frozen to a temperature of about −30° C. or lower (e.g., fromabout −30° C. to about −50° C.). More preferably, the liquid compositionis frozen to a temperature of about −45° C. or lower (e.g., from about−45° C. to about −60° C.). Most preferably, the liquid composition isfrozen to a temperature of about −47° C., −48° C., −49° C., or −50° C.

The composition can be frozen rapidly (e.g., by contacting a containerof the solution in a cooling bath), or by cooling in stages (e.g., bylowering the temperature incrementally at progressively lowertemperatures until the frozen mixture is obtained). Alternatively, thesterile solution can be frozen by continuously cooling at asubstantially constant rate until the frozen mixture is obtained. Forexample, the composition can be frozen by cooling at a substantiallyconstant rate of about 5° C. per minute or less (e.g., from about 0.1 toabout 5° C. per minute), at a rate of about 3° C. per minute or less(e.g., from about 0.1 to about 3° C. per minute), at a rate of about 2°C. per minute or less (e.g., from about 0.1 to about 2° C. per minute),or at a rate of about 1° C. per minute or less (e.g., from about 0.1 toabout 1° C. per minute, or from about 0.1 to about 0.5° C. per minute),until the frozen mixture is obtained. Most preferably, the compositionis frozen by cooling at a rate of about 0.1° C. per minute, about 0.2°C. per minute, about 0.3° C. per minute, about 0.4° C. per minute, orabout 0.5° C. per minute. Alternatively, the composition can be frozenusing a combination of incremental cooling stages and one or morecontinuous cooling cycles (e.g., continuously cooling at a substantiallyconstant rate) until the frozen mixture is obtained.

After the frozen mixture is obtained, the temperature can be maintainedfor any suitable length of time. Preferably, the temperature at whichthe composition is frozen is maintained for at least about 1 hour toabout 30 hours (e.g., about 1 hour, about 5 hours, about 15 hours, about25 hours, or about 30 hours). More preferably, the temperature at whichthe composition is frozen is maintained for at least about 10 hours toabout 20 hours (e.g., about 10 hours, about 12 hours, about 15 hours, orabout 19 hours). Most preferably, the temperature at which thecomposition is frozen is maintained for at least about 15 hours.

The primary drying temperature in the primary drying phase is preferablyfrom about −30° C. to about 35° C., especially from about −30° C. toabout 20° C., more preferably from about −15° C. to about 20° C.,desirably from about −15° C. to about 10° C., and still more desirablyfrom about −15° C. to about 0° C. Most preferably, the primary dryingtemperature is from about −15° C. to about −10° C. (e.g., is about −15°C.).

In the primary drying stage, the temperature of the frozen compositioncan be changed in stages (e.g., raised or lowered incrementally atprogressively higher or lower temperatures until the primary dryingtemperature is attained). Alternatively, the temperature of the frozencomposition in the primary drying stage can be changed continuously(e.g., raised or lowered at a substantially constant rate) until theprimary drying temperature is attained. Preferably, the temperature ofthe frozen composition in the primary drying stage is changed at a rateof about 5° C. per minute or less (e.g., from about 0.05° C. to about 2°C. per minute). More preferably, the temperature of the frozencomposition in the primary drying stage is changed at a rate of about 3°C. per minute or less (e.g., from about 0.05° C. to about 3° C. perminute). Still more preferably, the temperature of the frozencomposition in the primary drying stage is changed at a rate of about 2°C. per minute or less (e.g., from about 0.1° C. to about 2° C. perminute). Most preferably, the temperature of the frozen composition inthe primary drying stage is changed at a rate of about 1° C. per minuteor less (e.g., from about 0.1° C. to about 1° C. per minute, or fromabout 0.1° C. to about 0.5° C. per minute). In a particularly preferredembodiment, the temperature of the frozen composition in the primarydrying stage is changed at a rate of about 0.5° C. per minute or less(e.g., about 0.5° C. per minute, about 0.4° C. per minute, about 0.3° C.per minute, about 0.2° C. per minute, or about 0.1° C. per minute).

The primary drying temperature in the primary drying stage is preferablymaintained (e.g., held at a substantially constant temperature or keptwithin a particular range) until substantially all of the tert-butylalcohol solvent is removed. In this regard, the primary dryingtemperature desirably is maintained for at least about 40 hours to about80 hours (e.g., about 40 hours, about 50 hours, about 60 hours, about 70hours, or about 80 hours). Preferably, the primary drying temperature ismaintained for at least about 60 hours to 70 hours (e.g., about 62hours, about 65 hours, or about 67 hours). Most preferably, the primarydrying temperature is maintained for at least about 67 hours.

In general, as the temperature is raised during the primary dryingstage, the internal temperature “lags” behind (i.e., is lower than) theexternal temperature (sometimes referred to as the “shelf temperature”).In some instances when the external temperature is raised during theprimary drying stage, the internal temperature can lag behind theexternal temperature by as much as about 10° C., or even more.Typically, the removal of substantially all of the solvent can bedetermined by comparing the internal temperature with the externaltemperature. The temperature of the frozen mixture and the externaltemperature can be measured using any suitable means, e.g., athermometer, a thermocouple, or the like. In most instances,substantially all of the tert-butyl alcohol solvent is removed when theinternal temperature remains steady or is about equal to (e.g., isslightly less than, is equal to, or slightly exceeds) the externaltemperature. In a preferred embodiment, the primary drying temperatureis maintained until the temperature of the frozen mixture is about equalto the primary drying temperature.

The secondary drying temperature in the secondary drying stage can rangefrom about 0° C. to about 45° C., but is preferably from about 10° C. toabout 40° C. More preferably, the secondary drying temperature is aboutambient temperature (e.g., from about 20° C. to about 45° C., even morepreferably from about 30° C. to about 45° C.), and is most preferablyfrom about 20° C. to about 35° C. (e.g., is about 30° C.). In thesecondary drying stage, the temperature of the frozen composition can bechanged (e.g., raised or lowered) at a rate which is the same ordifferent than the rate at which the temperature is changed in theprimary drying stage. For example, the temperature of the frozencomposition in the secondary drying stage can be changed in stages(e.g., raised or lowered incrementally at progressively higher or lowertemperatures until the secondary drying temperature is attained).

Alternatively, the temperature of the frozen composition in thesecondary drying stage can be changed continuously (e.g., at asubstantially constant rate) until the secondary drying temperature isattained. Preferably, the temperature of the frozen composition in thesecondary drying stage is changed at a rate of about 5° C. per minute orless (e.g., from about 0.1° C. to about 5° C. per minute). Morepreferably, the temperature of the frozen composition in the secondarydrying stage is changed at a rate of about 3° C. per minute or less(e.g., from about 0.1° C. to about 3° C. per minute). Still morepreferably, the temperature of the frozen composition in the secondarydrying stage is changed at a rate of about 2° C. per minute or less(e.g., from about 0.1° C. to about 2° C. per minute). Most preferably,the temperature of the frozen composition in the secondary drying stageis changed at a rate of about 1° C. per minute or less (e.g., from about0.1° C. to about 1° C. per minute, or from about 0.1° C. to about 0.5°C. per minute). In a particularly preferred embodiment, the temperatureof the frozen composition in the secondary drying stage is changed at arate of about 0.5° C. per minute or less (e.g., about 0.5° C. perminute, about 0.4° C. per minute, about 0.3° C. per minute, about 0.2°C. per minute, or about 0.1° C. per minute).

Preferably, the secondary drying temperature in the secondary dryingstage is maintained until the moisture content is less than about 2.0 wt% (% liquid relative to the dry weight of the lyophilized tobramycinformulation). More preferably, the secondary drying temperature in thesecondary drying stage is held until the moisture content is about 1.0wt % or less. Most preferably, the secondary drying temperature in thesecondary drying stage is held until the moisture content is about 0.5wt % or less.

The secondary drying temperature preferably is maintained for at leastabout 5 hours, especially for from about 5 hours to about 30 hours(e.g., about 5 hours, about 15 hours, about 18 hours, about 20 hours,about 25 hours, or about 30 hours). More preferably, the secondary dyingtemperature is maintained for at least from about 10 hours to about 20hours (e.g., about 10 hours, about 12 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, or about 19 hours). Mostpreferably, the secondary drying temperature is maintained for about 15hours, about 16 hours, about 17 hours, about 17.5 hours, about 18 hours,about 18.5 hours, or about 19 hours.

The primary drying stage is preferably carried out at a pressure ofabout 500 micron Hg (about 67 Pascal (Pa) or less (e.g., from about 10micron Hg to about 500 micron Hg (or about 1 Pa to about 67 Pa)). Morepreferably, the pressure is about 300 micron Hg (about 40 Pa) or less(e.g., from about 10 micron Hg to about 300 micron Hg (or about 1 Pa toabout 40 Pa)). Most preferably, the primary drying stage is carried outat a pressure of about 150 micron Hg (or about 20 Pa) or less (e.g.,from about 10 micron Hg to about 150 micron Hg (or about 1 Pa to about20 Pa)). In a particularly preferred embodiment, the primary dryingstage is carried out at a pressure of about 100 micron Hg (about 13 Pa).

The secondary drying stage can be carried out at a pressure which is thesame or different than the pressure at which the primary drying stage iscarried out. Preferably, the secondary drying stage is carried out at apressure of about 500 micron Hg (about 67 Pa) or less (e.g., from about10 micron Hg to about 500 micron Hg (or about 1 Pa to about 67 Pa). Morepreferably, the pressure is about 300 micron Hg (or about 40 Pa) or less(e.g., from about 10 micron Hg to about 300 micron Hg (or about 1 Pa toabout 40 Pa)). Most preferably, the primary drying stage is carried outat a pressure of about 150 micron Hg (or about 20 Pa) or less (e.g.,from about 10 micron Hg to about 150 micron Hg (or about 1 Pa to about20 Pa)). In a particularly preferred embodiment, the secondary dryingstage is carried out at a pressure of about 100 micron Hg (or about 13Pa).

The invention further provides a pharmaceutical dosage form comprising asealed container and a pharmaceutical formulation comprising atherapeutically effective amount of lyophilized tobramycin containedwithin the container, wherein the lyophilized tobramycin is in the formof a free-flowing powder. Descriptions of the lyophilized tobramycin andfree-flowing powder, and components thereof, set forth above inconnection with the inventive pharmaceutical formulation, also areapplicable to those same aspects of the inventive pharmaceutical dosageform. The pharmaceutical dosage form can be a sterile single-dose orsterile multiple-dose dosage form. Exemplary pharmaceutical dosage formsinclude a pharmaceutical dosage form comprising a sealed container(e.g., a container as described herein) and the inventive pharmaceuticalformulation comprising a therapeutically effective amount of lyophilizedtobramycin contained within the container.

The inventive pharmaceutical dosage form preferably includes a dose oflyophilized tobramycin of from about 0.5 grams to about 5.0 grams (e.g.,about 1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 3.0 g, about3.5 g, about 4.0 g, about 4.5 g, or about 5.0 g) contained within thecontainer. Most preferably, the pharmaceutical dosage form includes adose of the lyophilized tobramycin of about 1.2 g contained within thecontainer.

To prepare the pharmaceutical dosage form, the pharmaceuticalformulation can be packaged in the container by any suitable methodknown in the art. In a preferred embodiment of the invention, theinventive pharmaceutical formulation is packaged in the container by amethod comprising the steps of (a) filling one or more containers with asterile liquid composition comprising a therapeutically effective amountof tobramycin (e.g. present as tobramycin sulfate) and a solventcomprising tert-butyl alcohol (e.g., a solvent comprising tert-butylalcohol and water), each container defining an opening, (b) subjectingthe composition in the one or more containers to the lyophilizedtobramycin production method described herein, and (c) sealing theopening of the one or more containers, to produce the pharmaceuticaldosage form.

The one or more containers preferably include one or more sterile vials,preferably glass vials, as described herein. The sealing step preferablyincludes sealing the opening using the means for aseptically sealing theopening described herein. The sealing means preferably includes astopper as described herein.

The sterile liquid composition added to one or more containers (prior tolyophilization) preferably contains tobramycin in an amount of fromabout 60 mg/mL to about 100 mg/mL, and even more preferably containstobramycin in an amount of from about 70 mg/mL to about 90 mg/mL (e.g.,about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, or about90 mg/mL). In a particularly preferred embodiment, the concentration ofthe sterile solution is about 83.2 mg/mL and the one or more containers(which are most preferably vials) are filled with 50 mL or less (e.g.,about 40 mL, about 30 mL, about 20 mL, about 10 mL, or about 5 mL) ofthe sterile solution, to provide a final dosage of about 1.2 g oftobramycin.

The above-described method of preparing the inventive pharmaceuticaldosage form can consistently and reproducibly produce dosage forms withhigh dosage accuracy and low variability in the dosage. Moreover, themethod is simpler and is significantly less costly than the conventionalmethods used in the production of tobramycin sulfate powder.

The pharmaceutical dosage form prepared in accordance with the presentinvention preferably is within about 20% of the label claim. In otherwords, the amount of tobramycin in the container (as determined by asuitable analytical technique, e.g., HPLC, tobramycin sulfate assay, orthe like) preferably is within about 20 wt % of the tobramycin dosageclaimed in the product label. Most preferably, the inventivepharmaceutical dosage form has an actual dosage of tobramycin that iswithin about 10% for its lower end and within about 15% for its higherend of the label claim. By way of example, for 1.2 g dosage vialsprepared in accordance with the present invention, with a label claim of1.2 g of tobramycin, the amount of tobramycin in the vials (e.g.,present as tobramycin sulfate), as determined by a suitable analyticaltechnique, preferably is within about 1.08 g to about 1.38 g. Even morepreferably, the inventive pharmaceutical dosage form has an actualdosage of tobramycin that is within about 10% of the label claim. Mostpreferably, the inventive pharmaceutical dosage form has an actualdosage that is within about 5%, about 4%, or about 3% of the labelclaim.

Also provided by the invention is a method of treating a disease in apatient in need thereof. The method comprises dissolving the inventivepharmaceutical formulation comprising lyophilized tobramycin infree-flowing powder form in a pharmaceutically acceptable solvent toproduce a pharmaceutically acceptable solution, and administering thesolution to the patient. The lyophilized tobramycin formulation can beadministered to a patient in need thereof (e.g., to treat microbialinfections) using standard therapeutic methods for deliveringtobramycin. While any suitable means of administering the pharmaceuticalformulation to a human can be used within the context of the invention,typically and preferably the inventive method of treating a disease in apatient involves administering the pharmaceutical formulation to a humanvia injection. By the term “injection,” it is meant that the compositionis forcefully introduced into a target tissue of the human. Thepharmaceutical formulation can be administered to the human by anysuitable route, but preferably is administered to the humanintravenously or intramuscularly. When the inventive composition isadministered by injecting, any suitable injection device can be used.While less preferred, other routes of administration can be used todeliver the pharmaceutical formulation to the human in accordance withthe inventive method. Indeed, although more than one route can be usedto administer the inventive formulation, a particular route can providea more immediate and more effective reaction than another route.

The inventive pharmaceutical formulation comprising lyophilizedtobramycin can be reconstituted for parenteral administration to apatient using any pharmaceutically acceptable diluent. Preferably, thediluent is Sterile Water for Injection, USP. Alternatively, the diluentmay be, for example, 5% or 10% Dextrose in water, USP, 0.9% SodiumChloride Injection, USP, 5% Dextrose and 0.9% Sodium Chloride, Ringer'sSolution, or Lactated Ringer's Injection, USP. Preferably thereconstitution is done in Sterile Water for Injection, USP, and thereconstituted solution optionally is further diluted, e.g., in 5%Dextrose in water, USP, or 0.9% Sodium Chloride Injection, USP.

Any quantity of diluent may be used to reconstitute the lyophilizedtobramycin such that a suitable solution for injection is prepared.Accordingly, the quantity of diluent must be sufficient to dissolve thelyophilized tobramycin. Typically, about 50 mL or less (e.g., about 40mL, about 30 mL, about 20 mL, about 10 mL, or about 5 mL) of diluent areused to reconstitute the lyophilized tobramycin to yield a finalconcentration of about 40 mg/mL or less (e.g., about 40 mg/mL, about 35mg/mL, about 30 mg/mL, about 25 mg/mL, about 20 mg/mL, about 15 mg/mL,or 10 mg/mL, about 5 mg/mL, or about 1 mg/mL). More preferably, thereconstituted solution is further diluted to a final concentration oftobramycin of about 2 mg/mL or less (e.g., about 1.5 mg/mL, about 1.0mg/mL, about 0.5 mg/mL, about 0.4 mg/mL, about 0.3 mg/mL, or about 0.2mg/mL). Most preferably, the final concentration of tobramycin is fromabout 0.2 mg/mL to about 2 mg/mL.

Prior to reconstitution, the inventive lyophilized tobramycin dosageform should be stored at controlled room temperature, preferably about59° F. to about 86° F. (15° C. to 30° C.). Reconstituted solutions oflyophilized tobramycin should be administered to a patient promptly uponconstitution. Alternatively, reconstituted solutions should berefrigerated and used within 96 hours. If kept at room temperature,reconstituted solutions of the pharmaceutical formulation should be usedwithin about 24 hours.

Solutions of tobramycin may be further diluted after reconstitutionusing any suitable diluent. Preferred for further dilution are 5%Dextrose in water, USP, or 0.9% Sodium Chloride Injection, USP. Otherfluids for further dilution of solutions of reconstituted lyophilizedtobramycin include, for example, 10% Dextrose in water, USP, 5% Dextroseand 0.9% Sodium Chloride, Ringer's Solution, Lactated Ringer'sInjection, USP, or Sterile Water for Injection, USP.

The inventive pharmaceutical formulation can be administered to apatient (e.g., a human patient) to treat or prevent any disease orcondition against which tobramycin (e.g., tobramycin sulfate) is active.In this regard, the pharmaceutical formulation can be administered to apatient suffering from, for example, septicemia, complicated andrecurrent urinary tract infections, lower respiratory infections,serious skin and soft tissue infections including burns and peritonitis,and central nervous system (CNS) infections caused by organismsresistant to other antibiotics.

In addition or alternatively, the inventive pharmaceutical compositioncan be administered to a patient that has been infected by amicroorganism that is sensitive to tobramycin. Such microorganismsinclude, for example, P. aeruginosa, Proteus sp. (indole-positive andindole-negative), including P. mirabilis, M. morganii, P. rettgeri, andP. vulgaris, E. coli, Klebsiella-Enterobacter-Serratia group,Citrobacter sp., Providencia sp., Staphylococci, including S. aureus(coagulase-positive and coagulase-negative). These microorganisms,however, are merely exemplary. Indeed, the inventive pharmaceuticalformulation can be administered to a patient that has been infected withany microoganism that is sensitive to (i.e., whose protein synthesis isirreversably inhibited by) tobramycin.

In addition to the preferred embodiments described herein, the inventivepharmaceutical formulation can comprise additional therapeutic orbiologically active agents. For example, therapeutic factors useful inthe treatment of a particular indication (e.g., septicemia) can bepresent. Factors that control inflammation, such as ibuprofen orsteroids, can be part of the composition to reduce swelling andinflammation associated with in vivo administration of the compositionand physiological distress. Immune enhancers can be included in thecomposition to up regulate the body's natural defenses against disease.Vitamins and minerals, antioxidants, and micronutrients can beco-administered with the composition.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the preparation of a liquid compositioncomprising tobramycin sulfate and a solvent containing tert-butylalcohol.

A quantity of sterile water for injection (“WFI”) was collected(approximately 80% of total batch quantity) and was placed into a clean,jacketed Type 316 stainless steel compounding tank. The WFI was at atemperature of about 15° C. to 30° C. Tobramycin sulfate, USP rawmaterial (obtained from a commercial supplier, 5.57 kg) was added to theWFI in the compounding tank. The resulting mixture was stirred at 716rpm until the tobramycin sulfate was completely dissolved in the WFI (atleast 10 minutes).

After the tobramycin was dissolved, the solution was stirred at 625 rpm,and 1.59 kg of tert-butyl alcohol, which had been warmed to atemperature of about 30° C. and 45° C., was added during stirring. Themixture was stirred at 719 rpm until the tert-butyl alcohol wascompletely dissolved (at least 10 minutes).

After the tert-butyl alcohol was dissolved, the batch volume was raisedto 45 L (total batch quantity) by the addition of WFI, USP, and thesolution was stirred at 714 rpm for at least an additional 10 minutes.

Prior to filtration, an in-process sample was taken from the batch tank,and was subjected to an appearance test (visual examination) andtobramycin sulfate assay. After completion of the in-process appearancetest and tobramycin sulfate assay, the cooled solution of tobramycinsulfate in WFI was pumped through a 0.45 μm pre-filter (Opticap® orDurapore® PVDF available from Millipore Corporation) into a filling roomusing Tygon® tubing. The pre-filter was rinsed with WFI, USP prior tofiltration of the tobramycin sulfate solution. Near the end of thefiltration step, a sample was collected and subjected to apre-filtration bioburden test to determine the action limit of thematerial, which should be not more than 10 CFU/mL.

Following pre-filtration, the tobramycin sulfate solution was passedthrough a sterile 0.22 μm final filter (Opticap® or Millidisk® PVDFavailable from Millipore Corporation) and the filtrate was deliveredinto a clean, sterile receiving carboy using silicone tubing. In a class100 clean room environment, sterile vials were aseptically filled withthe solution and partially stoppered.

EXAMPLE 2

This example demonstrates a method of lyophilizing a pharmaceuticalformulation comprising tobramycin sulfate.

A nitrogen supply was connected to a sterilizing filter assembly on alyophilizer and the lyophilizer chamber and condenser were steamsterilized for at least 30 minutes using standard sterilizationprocedures. The minimum chamber drain temperature and minimum condenserdrain temperature were each at least 121.0° C. After sterilization, avessel integrity test was performed. The shelves and condenser plates ofthe lyophilizer were chilled to −30° C. and −50° C., respectively, andthe vessel was leak tested.

The lyophilization process was initiated by pre-chilling the shelves ofthe lyophilizer. The shelf temperature controller was adjusted to a setpoint of 5° C. After the pre-chill set point was reached, the shelveswere loaded with vials containing a tobramycin sulfate compositionprepared as described in Example 1 over approximately 8 hours. After theproduct solution was completely loaded, the chamber door was closed andthe shelf temperature was maintained at the pre-chill set point for atleast 60 minutes. The shelf temperature controller was then adjusted toa set point of −50° C. with a ramp time of 265 minutes and the shelftemperature was held at the set point temperature of −50° C. for atleast 15 hours.

When the condenser temperature reached −48° C., the primary drying stepwas performed. The vacuum controller set point was set to 100 μm Hg (13Pa) and the vacuum alarm high set point was set to 125 μm (16 Pa).Nitrogen gas was used to regulate the pressure. The shelf temperaturecontroller was adjusted to a primary drying set point of −15° C. with aramp time of 5.5 hours and the shelf temperature was held at the dryingset point temperature of −15° C. for 67 hours.

The shelf temperature controller was adjusted to a secondary drying setpoint of 30° C. with a ramp time of 225 minutes and the shelftemperature was held at the secondary drying set point temperature of30° C. for at least 18 hours. At the end of the 18-hour secondary dryinghold time, the chamber was isolated and the vacuum was released. Thepressure was raised to atmospheric pressure by addition of sterilenitrogen gas.

The vials containing the lyophilized tobramycin sulfate were thenstoppered with Stelmi 6720GC Gray Lyo stoppers using an internalstoppering mechanism. At the completion of the stoppering step, thechamber was again isolated and the vacuum released. The pressure wasraised to atmospheric pressure by addition of sterile nitrogen gas. Thelyophilizer chamber was unloaded and the stoppered vials containinglyophilized product were conveyed to a capping machine and sealed withaluminum seals. The vials were then inspected, labeled, and packaged.

The final product was a sterile, white to off-white solid having greaterthan 98% purity and was suitable for administration by injection.Solutions prepared by dissolving the final product in Sterile Water forInjection were clear and free of particulates.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of producing a stable, sterile pharmaceutical formulationcomprising lyophilized tobramycin, which method comprises: (a) preparinga liquid composition comprising tobramycin and a solvent comprisingabout 4.5% by volume or less of tert-butyl alcohol, (b) freezing thecomposition to a temperature of from about −10° C. to about −70° C., toproduce a frozen mixture, wherein the temperature is maintained for fromat least about 1 hour to about 30 hours, (c) subjecting the frozenmixture to a primary drying stage, which comprises applying a vacuum toreduce the pressure to about 100 micron Hg, and, while applying thevacuum, changing the temperature of the frozen mixture to a primarydrying temperature of from about −15° C. to about 20° C., wherein thetemperature of the frozen mixture in the primary drying stage is changedat a rate of from about 0.1° C. to about 0.5° C. per minute, and whereinthe primary drying temperature is maintained for at least about 40 hoursto about 80 hours, to produce a first intermediate, and (d) subjectingthe first intermediate to a secondary drying stage, which comprisesapplying a vacuum to reduce the pressure to about 100 micron Hg, and,while applying the vacuum, changing the temperature of the frozenmixture to a secondary drying temperature of from about 30° C. to about45° C., wherein the temperature of the frozen mixture in the secondarydrying stage is changed at a rate of from about 0.1° C. to about 0.5° C.per minute, and wherein the secondary drying temperature is maintainedfor at least about 5 hours to about 30 hours, to produce thepharmaceutical formulation, wherein the lyophilized tobramycin is in theform of a free-flowing powder.
 2. The method of claim 1, wherein thecomposition is frozen to a temperature of from about −45° C. to about−60° C.
 3. The method of claim 1, wherein the composition is frozen to atemperature of about −45° C.
 4. The method of claim 1, wherein theprimary drying temperature is from about −15° C. to about 10° C.
 5. Themethod of claim 1, wherein the temperature at which the composition isfrozen is held for at least about 10 hours to about 20 hours.
 6. Themethod of claim 1, wherein the temperature of the frozen mixture in theprimary drying stage is raised at a rate of about 0.1° C. per minute. 7.The method of claim 1, wherein the primary drying temperature in theprimary drying stage is maintained for at least about 60 hours to about70 hours.
 8. The method of claim 1, wherein the primary dryingtemperature in the primary drying stage is maintained until thetemperature of the frozen mixture is about equal to the primary dryingtemperature.
 9. The method of claim 1, wherein the secondary dryingtemperature is from about 30° C. to about 40° C.
 10. The method of claim1, wherein the temperature of the frozen mixture in the secondary dryingstage is raised at a rate of about 0.2° C. per minute.
 11. The method ofclaim 1, wherein the secondary drying temperature in the secondarydrying stage is maintained for at least about 10 hours to about 20hours.
 12. The method of claim 1, wherein the secondary dryingtemperature in the secondary drying stage is held until the moisturecontent is about 1.0 wt % of the formulation.
 13. The method of claim 1,wherein (a) the primary drying temperature is from about −15° C. toabout −10° C., (b) the temperature of the frozen mixture in the primarydrying stage is raised at a rate of about 0.1° C. per minute, and (c)the primary drying temperature is maintained for at least about 60 hoursto about 70 hours.
 14. The method of claim 1, wherein (a) thecomposition is frozen to a temperature of from about −45° C. to about−60° C., and the temperature at which the composition is frozen ismaintained for at least about 10 hours to 20 hours, (b) the primarydrying temperature is from about −15° C. to about −10° C., and theprimary drying temperature is maintained for at least about 60 hours toabout 70 hours, and (c) the secondary drying temperature is from about30° C. to about 40° C., and the secondary drying temperature ismaintained for at least about 10 hours to about 20 hours.
 15. The methodof claim 1, wherein (a) the composition is frozen to a temperature ofabout −48° C., and the temperature at which the composition is frozen ismaintained for at least about 15 hours, (b) the primary dryingtemperature is about −15° C., and the primary drying temperature ismaintained for at least about 67 hours, and (c) the secondary dryingtemperature is about 30° C., and the secondary drying temperature ismaintained for at least about 15 hours.